The present invention concerns a new kit for detecting or quantifying the amount of one or multiple nucleic acid targets in a biological sample, and a detection or quantification method using this kit.
MicroRNAs are a kind of single-strand nucleic acid of about 22 nucleotides. Most of microRNAs are involved in tissue-specific gene expression control by acting as post-transcriptional repressors through binding to 3′-untranslated regions of target messenger RNA. More than half of the human genes are regulated by one or more microRNAs (either same or different type) and one type of microRNA can have several hundreds of target genes. Physiological homeostasis of cells and tissues is maintained by complex regulation, and therefore microRNA dysregulation can lead to diseases such as cancer or cardiovascular diseases. Due to their importance in disease development, microRNAs have become next generation biomarkers for diagnosis and prognosis of such diseases. As peripheral blood contains many different nucleic acids, the sensitive detection of circulating microRNA in plasma or serum can provide easily accessible genetic information.
There is a strong demand for routine quantitative profiling of microRNA dysregulations by measuring total RNA extracts from blood and tissues or by direct serum or whole blood analysis. Northern blot analysis, microRNA microarrays, and stem-loop primer-based quantitative real-time PCR are the major clinical research techniques for the search and identification of microRNAs as biomarkers for diseases.
Various other technologies for direct and sensitive detection of microRNA have been developed, including electrochemistry, surface enhanced Raman spectroscopy (Abell et al., 2012), surface plasmon resonance ({hacek over (S)}ípová et al., 2010), total internal reflection ellipsometry (Le et al., 2012), quartz crystal microbalance (Chen et al., 2009), and nanoparticle, nanowire (Zhang et al., 2009), and nanopore based technologies (Jain et al., 2013). Although some of these techniques can even distinguish single base pair mismatches under idealized experimental conditions, a common drawback is lacking applicability for real-life clinical diagnostics. In such a realistic detection scenario, an ideal microRNA biosensor must be able to distinguish multiple unique sequences as well as highly similar microRNAs in a complex sample, containing many different microRNAs (and precursor microRNAs) at different concentrations. Biofouling problems of solid supports, low throughput, difficulty in upscaling, and time-consuming and labor-intensive washing steps further limit the application of these technologies as standard tools in clinical diagnostics. A commercial technology for multiplexed microRNA (and other RNAs) detection using fluorescence barcoding is “Nanostring” (Kulkarni et al., 2001). However, this heterogeneous technology requires long incubation and separation steps and dedicated equipment, which are two of the main drawbacks of this technology for easily applicable clinical diagnostics.
WO2005/103298 describes a method for the simultaneous detection of a plurality of distinct target non-coding RNAs, such as microRNA or siRNA. Said method uses, for each target nucleic acid, (i) a first probe comprising a complementary nucleic acid of said target nucleic acid and a first signal generator to generate a first detectable signal and (ii) a second probe comprising a complementary nucleic acid of said target nucleic acid and a second signal generator to generate a second detectable signal. The presence of said plurality of target non-coding RNAs is determined by measuring the first and second detectable signal.
US2011/0033855 provides a similar method for quantifying nucleic acid molecules, such as microRNA, siRNA, in a nucleic acid-containing sample by utilizing fluorescence resonance energy transfer and a photocrosslinking reaction. Said method uses a first nucleic acid molecule probe comprising a sequence complementary to the target nucleic acid molecule and conjugated with a first marker, and a second nucleic acid molecule probe comprising a sequence complementary to the first nucleic acid molecule probe and conjugated with a second marker. However, this method does not overcome the drawback of prior art.
In fact, the short lengths (approximately 22 nucleotides) and strong sequence similarities are disadvantageous for the development of specific and sensitive microRNA detection technologies. Large melting temperature difference due to the sequence variability among microRNAs have made hybridization-based capturing probes difficult to apply for large scale expression profiling (de Planell-Saguer & Rodicio, 2011). Practical issues of pre-amplification in clinical setups, the requirement of microRNA labelling or indirect amplification, and complementary DNA conversion steps (often the primary cause of variations) are among the main problems for establishing the standard technologies as robust tools for real-life clinical diagnostics (Gao et al., 2013).
Consequently, there is still a great need to develop specific and efficient assays for detecting or quantifying short nucleic acids, with labelled probes which are homogeneous (e.g., no washing and separation steps), sensitive (detection of clinically relevant concentrations), specific (e.g., discrimination of very similar sequences in a complex mixture), fast (liquid phase binding kinetics and quick measurement), reproducible (ratiometric measurement), robust (stable sensing properties of bioconjugates), storable (e.g., stable lyophilized bioconjugates within an assay kit format), versatile (generic format for many microRNAs, facile bioconjugate production/purification), and multiplexed (simultaneous measurement of several microRNAs).